The protein tyrosine phosphatase superfamily, all of which contain a highly conserved active site motif, Cys-X5-Arg (CX5R), are key mediators of a wide variety of cellular processes, including growth, metabolism, differentiation, motility, and programmed cell death. Our laboratory has demonstrated that a subset of phosphatases harboring CX5R motifs utilize phosphoinositides instead of phosphoproteins as their physiological substrates. This includes the myotubularin related (MTMR) subfamily that removes the 3-phosphate from phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylolinositol 3,5-bisphosphate (PI(3,5)P2). Mutations in three different MTMR family members have been shown to cause the human neuromuscular disorders, myotubular myopathy and Charcot-Marie-Tooth disease. This proposal will study the structure, function, and regulation of the MTMR family of PTPs, as well as two novel lipid phosphatases, Jumpy and PLIP. Specific Aim 1 will determine the structural basis of how MTMR2 performs its biological function. In Specific aim 1A, the X-ray crystal structure of a Cys/Ser mutant of MTMR2 will be solved in a complex with its phosphoinositide substrate. This will provide the first structure of this family of phosphoinositide phosphatases in which the enzyme is complexed to its substrate. In Specific Aim 1B, we will delineate the structural changes, i.e. the conformational states and dynamics, that accompany substrate binding using deuterium exchange mass spectrometry (DXMS). Together, these studies will provide a comprehensive profile of how substrate specificity is determined for these enzymes as well as how phospholipid and other interactions influence enzyme activity. Specific Aim 2 addresses the mechanism(s) of MTMR2 and MTMR13[sbf2] regulation through protein-protein interaction. Specifically, the effects of the interaction of the catalytically inactive MTMR13[sbf2] with MTMR2 will be studied biochemically and transgenic mouse models will be established by "knocking out" MTMR2 and MTMR13[sbf2]. These studies will provide key insights into how the active and inactive MTMRs interact and the effects of their interactions on cellular phospholipid levels. It will also specifically address the roles that MTMR2 and MTMR13[sbf2] play in Charcot- Marie-Tooth disease. In Specific Aims 3 and 4, two novel PTP-like lipid phosphatases, Jumpy and PLIP, will be characterized with respect to cellular substrates and function. Jumpy is a 3-phosphoinositide phosphatase that is linked to a characteristic muscle phenotype in flies, and PLIP is a putative phosphatidylinositol 5-phosphate (PI(5)P) phosphatase. PI(5)P is a phospholipid of previously unrecognized importance that may be involved in several cellular paradigms including tumor suppression. Collectively, these results will provide a strong basis for understanding the cellular functions and regulations of the MTMR family as well as two new phospholipid phosphatases.